This invention relates to gene therapy to cause expression of genes within the epidermis or epidermal cells.
The skin is the largest organ in the human body. The skin consists of two layers, the epidermis and the dermis. The outer layer is the epidermis which is composed of four histologically defined layers, each of which represent a distinct stage of differentiation of the epidermal keratinocyte. The innermost layer is the stratum germinativum (or basal layer) consisting of continuously dividing cells. The next two layers are the stratum spinosum (or spinous layer) and the stratum granulosum (or granular layer). The outermost layer is the stratum corneum consisting of dead cells whose cytoplasm has been entirely replaced by keratin. (Iverson, et al., Cell Tissue Kinet., Vol. 1, pp. 351-367 (1968); MacKenzie, et al., Nature, Vol. 226, pp. 653-655 (1970)).
The dermis lies under the epidermis and is separated from it by a basement membrane. The dermis is a thick layer of living tissue consisting mainly of a loose connective tissue within which are blood capillaries, lymph vessels, sensory nerve endings, sweat glands and their ducts, hair follicles, sebaceous gland and smooth muscle fibers.
The epidermis is a continuously regenerating epithelium. Keratinocytes are the major cell type of the epidermis and arise from the basal cells in the basal layer. The basal cells consist of metabolically active cells. The basal cells are the cells which undergo mitosis. (Potten, In Stem Cells: Their Identification and Characterization, pp. 200-232 (1983)).
Upon commitment to differentiation, the basal cells lose their proliferative potential and migrate to the spinous layer. With further maturation these cells enter the granular layer and finally terminate as cornified squames in the stratum corneum before being sloughed into the environment. (Matoltsky, J. Invest. Dermatol., Vol 65, pp. 127-142 (1975)).
During the regeneration process for the differentiated epidermal cells, the cells express a succession of different homologous keratin genes. The keratin produced by the differentiating cells is an insoluble fibrous protein. Keratins are the most abundant proteins synthesized in the epidermal cells and changes in the keratin expression patterns occur during differentiation.
The degree of differentiation can be defined biochemically by the expression of marker proteins that characterize each stage. (Matoltsky, J. Invest. Dermatol., Vol. 65, pp. 127-42 (1975)). For instance, basal keratinocytes express keratins K5 and K14 as major products. (Woodcock-Mitchell, et al., J. Cell Biol., Vol. 95, pp. 580-88 (1982)). These proteins assemble into 10 nm filaments and together with microtubules and microfilaments, comprise the cytoskeleton of epidermal cells. (Steinert, P. M., et al., Cell, Vol. 42, pp. 411-19 (1985)).
One of the earliest changes associated with the commitment to differentiation and migration into the spinous layer is the induction of another differentiation-specific pair of keratins, K1 and K10. Once a cell is committed to the differentiation pathway the cells downregulate the genes for K5 and K14, and express the genes for the differentiation-specific keratins, K1 and K10. (Woodcock-Mitchell, et al., J. Cell Biol., Vol. 95, pp. 580-88 (1982); Roop, et al., Proc. Natl. Acad. Sci., USA, Vol. 80, pp. 716-20 (1983); Schweizer, et al., Cell, Vol. 37, pp. 159-170 (1984)). Transcription of K1 and K10 is restricted to the spinous layer cells. The expression of K1 precedes K10 and is one of the earliest in keratinocyte differentiation. Occasionally, K1 can be observed in a basal cell that has already ceased mitotic activity and is about to migrate into the spinous layer. Haitfeld et al. Carcinogenesis, 12 pp. 2063-2067 (1991). When the cells mature into granular layer cells, the genes for K1 and K10 are downregulated. At this point, other genes, notably loricrin and filaggrin, are induced. (Dale, B. A., et al., Nature, Vol. 276, pp. 729-731 (1978); Harding, C. R., et al., J. Mol. Biol., Vol. 170, pp. 651-673 (1983)).
Genes or cDNAs encoding the major keratins expressed in epidermal cells have been cloned, such as K5 (Lersch, et al., Mol. and Cell Biol., Vol. 8, pp. 486-493 (1988)), K14 (Marchuk, et al., Proc. Natl. Acad. Sci., USA, Vol. 82, pp. 1609-1613 (1985); Knapp, et al., J. Biol. Chem., Vol. 262, pp. 938-945 (1987); Roop, et al., Cancer Res., Vol. 48, pp. 3245-3252 (1988)), K1 (Steinert, et al., J. Biol. Chem., Vol. 260, pp. 7142-7149 (1985)), and K10 (Krieg, et al., J. Biol. Chem., Vol. 260, pp. 5867-5870 (1985)). In addition, human K6 cDNA has been cloned. (Tyner, et al., Proc. Natl. Acad. Sci., USA, Vol. 82, pp. 4683-4687 (1985)).
Northern blot analysis and in situ hybridization studies suggest that keratin genes K5 and K14 are predominantly transcribed in the proliferating basal layer. Transcription of keratin genes K1 and K10 is induced as cells migrate into the spinous layer. (Lersch, et al., Mol. and Cell Biol., Vol. 8, pp. 486-493 (1988); Knapp, et al., J. Biol. Chem., Vol. 262, pp. 938-945 (1987); Roop, et al., Cancer Res., Vol. 48, pp. 3245-3252 (1988)). K6 is expressed in human skin under conditions of high proliferation and malignant transformation. (Tyner, et al., J. Cell Biol., Vol. 103, pp. 1945-1955 (1986)).
Genes encoding rat and mouse filaggrin have also been identified. In situ hybridization experiments confirmed that transcription of this gene is restricted to the granular layer. (Haydock, et al., J. Biol. Chem., Vol. 261, pp. 12520-12525 (1986); Rothnagel, et al., J. Biol. Chem., Vol. 262, pp. 15643-15648 (1987); Fisher, et al., J. Invest. Dermatol., Vol. 88, pp. 661-664 (1987)).
Loricrin, one of the genes encoding a component of a cornified envelope, has been studied at the molecular level by in situ hybridization showing that transcripts of this gene are restricted to the granular layer. (Mehrel, et al., Cell, Vol. 61, pp. 1103-1112 (1990)). Both the human loricrin gene (Yoneda, et al., J. Biol. Chem., Vol. 267, no. 25, pp. 18060-18066 (1992)), and the mouse loricrin cDNA (Mehrel, et al., Cell, Vol. 61, pp. 1103-1112 (1990)) have been isolated and cloned.
Studies have shown that cells generated by cultivation of a small biopsy can be prepared as stratified sheets and then used for replacement of damaged skin by grafting techniques. (Lindahl, et al., Growth Factors in Health and Disease, p. 388 (1990)). Other studies describe genetically engineered keratinocytes which synthesize human growth hormone. (Morgan, et al., Science, Vol. 237, pp. 1476-1479 (1987)). These studies described retrovirus mediated gene transfer to introduce recombinant human growth hormone into cultured human keratinocytes. The retroviruses were generated from the .PSI. AM cell line using an SV40 promoter. (Morgan, et al., Science, Vol. 237, pp. 1476-1479 (1987); Teumer, et al., Growth Hormone and Athymic Mice, FASEB, Vol. 4, pp. 3245-3250 (1990)). The transduced keratinocyte cultures secreted human growth hormone.
In addition, other studies have shown human keratinocytes permanently transformed with plasmids containing the human growth hormone gene under the control of either the metallothionein promoter or the herpesvirus thymidine kinase promoter. (Lindahl, et al., Growth Factors in Health and Disease, p. 388 (1990)). These studies also described skin grafting techniques with the genetically engineered keratinocytes.